ProjectSummary Over2millionpeoplesufferaseriousbacterialinfectioninvolvingantibioticresistanceeveryyearandatleast 23,000dieasadirectresult.Beta-lactamaseenzymescontributetothisresistancebyhydrolyzingantibiotics thatwouldnormallykillbacteria.Carbapenemases,aclassofbeta-lactamaseenzymes,areofparticular concernduetotheirabilitytobreakdownlast-resortantibiotics,carbapenems.Atpresent,thedeterminants thatcontributetothecarbapenemspecificityofthewidespreadKPCfamilyareunknown.KPC-2,a carbapenemase,andCTX-M9,anon-carbapenemase,offeranexemplarypairfortheanalysisofthese determinants.Theiroverallstructuralandmechanisticsimilaritiesallowforthefocusedinvestigationofhowthe 125positionalresiduedifferencesbetweentheseenzymesaltercarbapenemasespecificity.Weproposeto predictandidentifythesubsetsofthese125residuesthatarerequiredforcarbapenemasespecificityinKPC-2 andwouldgrantcarbapenemasespecificitytoCTX-M9.Wewillidentifythesesubsetsthroughtwoconcurrent approaches.Thefirstusesphylogeneticancestralmodelingtocreateanancestralcarbapenemaseanda closelyrelatedancestralnon-carbapenemase.Thisphylogeneticapproachallowsustoinfertwoancestorsof KPC-2andCTX-M9withdifferentcarbapenemasespecificities.Wewillthenusetheseenzymestounderstand thedeterminantsrequiredforcarbapenemasespecificityinasmallermutationalspaceascomparedtotheone forKPC-2andCTX-M9.Thesecondapproachinvolvestheuseofmoleculardynamicssimulationsand sequenceanalysisofKPC-2andCTX-M9toidentifyresiduepositionsmostlikelytoaccountforthisdifferent specificity.Inbothapproaches,wewillcreatemutantvariantsofenzymesthroughmutagenesisassaysand testactivitythroughantibioticresistanceandenzymeassays.Thestudyoftheresiduescontributingto carbapenemaseactivitywillofferinsightintothefunctionaldeterminantsunderlyingthesesimilarenzymesand providenewmethodsofantibioticresistancepredictioninclinicalandpharmaceuticalenvironments.